Interaction of the cation-dependent mannose 6-phosphate receptor with GGA proteins
Interaction of the cation-dependent mannose 6-phosphate receptor with GGA proteins
复制标题
DOI：
10.1074/jbc.m201879200
复制
发表时间：
2002-05-24
影响因子：
4.8
通讯作者：
Kornfeld, S
中科院分区：
文献类型：
--
作者：
Doray, B;Bruns, K;Kornfeld, S
高尔基体定位蛋白(γ)是一个多结构域的蛋白质家族，参与高尔基体和内体之间的蛋白质运输。最近的证据表明，阳离子非依赖型和阳离子依赖型甘露糖6-磷酸受体(MPR)通过细胞质尾部羧基末端的酸性簇-二亮氨酸基序与GGA的VHS结构域特异结合。然而，CD-MPR与VHS结构域的结合比CI-MPR更弱。对两个受体的C-末端残基的比对表明，酸性簇-二亮氨酸基序和侧翼残基存在一些非保守的差异。通过体外谷胱甘肽S转移酶下拉试验，CD-MPR胞质尾部的这些残基突变为CI-MPR的相应残基，可与GGA完全结合(H63D突变)、中间结合(R60S)或不变结合(E56F/S57H)。此外，CD-MPR的C-末端蛋氨酸抑制GGA结合，而CI-MPR的C-末端不抑制GGA结合。在Cl-MPR的C-末端Valine上添加4个丙氨酸也严重减少了GGA结合，表明酸性簇-二亮氨酸基序相对于C末端的间距对于最佳GGA相互作用是重要的。稳定表达CD-MPR的突变能增强GGA结合的小鼠L细胞比野生型CD-MPR更有效地分离组织蛋白酶D。这些研究为观察到的两个MPR对GGA蛋白的相对亲和力的差异提供了解释。此外，它们还表明GGA参与了CD-MPR介导的溶酶体酶分选。
The GGAs (Golgi-localizing, (γ) under bar -adaptin ear homology domain, ARF-binding) are a multidomain family of proteins implicated in protein trafficking between the Golgi and endosomes. Recent evidence has established that the cation-independent (Cl) and cation-dependent (CD) mannose 6-phosphate receptors (MPRs) bind specifically to the VHS domains of the GGAs through acidic cluster-dileucine motifs at the carboxyl ends of their cytoplasmic tails. However, the CD-MPR binds the VHS domains more weakly than the CI-MPR. Alignment of the C-terminal residues of the two receptors revealed a number of non-conservative differences in the acidic cluster-dileucine motifs and the flanking residues. Mutation of these residues in the CD-MPR cytoplasmic tail to the corresponding residues in the CI-MPR conferred either full binding (H63D mutant), intermediate binding (R60S), or unchanged binding (E56F/S57H) to the GGAs as determined by in vitro glutathione S-transferase pull-down assays. Furthermore, the C-terminal methionine of the CD-MPR, but not the C-terminal valine of the CI-MPR, inhibited GGA binding. Addition of four alanines to the C-terminal valine of the Cl-MPR also severely reduced GGA binding, demonstrating the importance of the spacing of the acidic cluster-dileucine motif relative to the C terminus for optimal GGA interaction. Mouse L cells stably expressing CD-MPRs with mutations that enhance GGA binding sorted cathepsin D more efficiently than wild-type CD-MPR. These studies provide an explanation for the observed differences in the relative affinities of the two MPRs for the GGA proteins. Furthermore, they indicate that the GGAs participate in lysosomal enzyme sorting mediated by the CD-MPR.